Modern biochemical assays used in human and veterinary medicine, research, and industry, are very often based on an interaction between a protein and its biological partner. If the protein is an antibody and the analyte to be determined is its corresponding antigen, the assays are commonly called immunotests or immunoassays. In the conventional arrangement, the protein is anchored to a solid surface (for example a plate or a bead) which enables the manipulation after the analyte had been bound to the protein. The beads with protein-analyte complex can be removed from the solution of a mixed sample, or the residual sample can be washed out from the plate before the detection, while the analyte remains anchored on the surface due to the interaction with the protein. The main advantage of this type of assays is their high specificity and enrichment of determined analyte which considerably helps enhancing the limit of detection and limit of quantification.
A wide variety of methods and techniques can be used for determination of an analyte enriched by means of an interaction with a protein. One of the key requirements is the compatibility with the used surface that serves as a substrate for the analysis. Desorption-ionization mass spectrometry is an important alternative to the current methods of detection, such as chemiluminescence, fluorescence, or radiation. In this technique, the analyte is desorbed from the surface while it is simultaneously ionized, and it is subsequently introduced to the mass analyzer and detected. Desorption ionization is usually carried out by a laser beam, but charged or neutral particles beam, plasma or electric discharge, electric field, stream of hot solvent steam, or beam of charged solvent droplets can be used instead. Compare to prevalent fluorescence or radiation detection the desorption-ionization mass spectrometry has the advantage of high specificity and possibility to get more information about the detected ion obtained by ionization of the analyte molecule than the conventional detection techniques.
The first technology based on modified surfaces for the desorption-ionization mass spectrometry was named SELDI (Surface-Enhanced Laser Desorption/Ionization) (Hutchens T W and Yip T T. “New desorption strategies for the mass spectrometric analysis of macromolecules.” Rapid Commun Mass Spectrom 7: 576-580 (1993). The less specific SELDI surfaces that bind the whole groups of analytes were developed, with different organic materials on the surface ((a) Tang N, Tornatore P, Weinberger S R (2004). “Current developments in SELDI affinity technology”. Mass spectrometry reviews 23 (1): 34-44; (b) Law K. P, Larkin S. R.: Anal Bioanal Chem (2011) 399:2597-2622), under many trade names (for example CM10—weak ion-exchange resin, Q10—strong ion-exchange resin, H50—hydrophobic reverse chromatography surface, IMAC30—phosphopeptides binding surface). More specific SELDI surfaces with bound proteins, antibodies included, are based on the usage of gold layer that is able to bind a wide variety of biological molecules due to the gold reactivity (U.S. Pat. No. 6,579,719; U.S. Pat. No. 6,844,165; U.S. Pat. No. 6,881,586). This process has the disadvantage of nonspecific bonding of all proteins in a sample onto the primary gold surface. This leads to reduction of detection specificity for the particular analyte, because not only the appropriate partner of an anchored protein binds onto the surface, but also many other molecules that can react with the gold surface. Alternatively, a molecule can be attached to a surface with the use of surface derivatization, for example by N-hydroxysuccinimide that can form the bond with a biological molecule via any nonprotected amine group (US20070059769 A1, WO2005088310A2). Substrate modification with phenylboronic acid, carboxyarylboronic acid, or aminophenylboronic acid is another chemical modification that enables binding of proteins. The surfaces modified in this way can be used as substrates for immunoassays as well (Liu and Scouten, J Mol Recognit. 1996 September-December; 9(5-6):462-7; Lee et al, S Am Soc Mass Spectrom. 2005 September; 16(9):1456-60, Farah et al, Biochim Biophys Acta. 2005 Oct. 10; 1725(3):269-82). The surfaces modified with phenylboronic acid or carboxyarylboronic acid were used for binding the glycosilized proteins from a sample, followed by desorption-ionization mass spectrometry detection, more particularly MALDI-MS (Gontarev et al. Rapid Communications in Mass Spectrometry 2007, 21(1): 1-6 and Shmanai Journal of Mass Spectrometry 2007 November, 42 (11): 1504-1513 and US 20080116367 A1). The coupling with the desorption-ionization mass spectrometry can be performed better with the use of techniques that enable binding the protein directly onto a metallic surface (the substrate for mass spectrometry measurement). The reason is that bare metal surface with no chemical interlayer, provides better charge transfer and thus more efficient ionization. However, most metals (for example stainless steel or aluminium) cannot be modified directly with proteins because no surface reaction occurs under normal conditions in solution. (Volny M. et al. Anal Chem 2005 August; 77(15):4890-6) used the process called reactive landing for modification of the stainless steel surfaces with proteins. This method of surface treatment was performed under vacuum and the plasma pre-treated surface was exposed to bombardment by desolvated ions; these ions were obtained by soft electrospray ionization of corresponding protein solutions. In the process of reactive landing according to Volny et al. (Volny M. et al. Anal Chem 2005 August; 77(15):4890-6), the charged protein ions, that hit the surface to be modified, translated in the vacuum environment with the mean free path sufficient to allow acceleration of the charged protein ions by external electrostatic field (up to 20V) and thus to reach considerably hyperthermal energy. At the time of impact, 20V of acceleration corresponds to more than thousand fold of average translational kinetic energy at the room temperature (according to the approximation based on kinetic theory of gases). Thus, the surface is modified due to the ion energy at the moment of ion impact on the surface—and therefore it is a kinetic energy induced reaction. The process provides high quality and mechanically stable surface modification. This method has the disadvantage of excessive length of the whole process of surface preparation, up to a couple of hours, and small efficiency of the transfer into the vacuum that results in high consumption of protein solution. Furthermore, to perform this process, it is necessary to design a special apparatus into which the surface to be modified is inserted. The apparatus is then evacuated and the vacuum has to be maintained during the whole time of the modification process. After the vacuum is reached, the hyperthermal ions are collision-landed onto the surface for approximately three hours. Thus, only a few samples a day can be prepared in the apparatus and the cost of one surface modification reflects that.
In PCT/CZ2011/000118 (WO/2012/079549); and Krasny et al. J Mass Spectrom 2012 October; 47(10):1294-302, a method for surface modification of substrates for determination of phosphopeptides by desorption-ionization mass spectrometry is described. The surface of the substrate is modified by the solution of organometallic compounds (organic propoxides) of the elements of group 4B, namely Ti, Zr, and Hf, by electrospraying. Organic propoxides are thermally decomposed and a chemical reaction proceeds, during which an appropriate propoxide transforms into an oxide that binds onto the surface. In this document, an inorganic surface is used to detect the analytes-phosphopeptides, i.e. structural elements of phosphorylated proteins. Decomposition reaction of proteins is undesirable, because it would lead to lost of their biological activity.
The overview documents (Jaworek et al. J Mater Sci (2007) 42:266-297 and Morozov Adv. Biochem Eng. Biotechnol. 2010, 119: 115-1620) describe electrospray droplets depositions where charged droplets or microdroplets impact on surfaces. However, these techniques cannot be used for modification of hard surfaces, such as metal surface. Furthermore, the electrospray deposition of proteins from charged droplets does not form stable enough protein layer on the surface. Thus, it requires further treatment, for example fixation with 70% solution of glutaraldehyde (Lee et al. Biomaterials 24 (2003) 2045-2051). Adjuvants, for example disaccharides, can be added to maintain the proteins' activity during their electrospray deposition (Morozov Anal. Chem., 1999, 71 (7), pp 1415-1420).